Development and validation of a LC-MS/MS method for the simultaneous analysis of diabetogenic lipids, diacylglycerols and ceramides, in subcellular fractions of skeletal muscle

Obesity presents a major public health challenge and is characterized by the excessive accumulation of lipids in ectopic tissue, such as liver and muscle. Specific lipid species, diacylglycerols (DAGs) and ceramides (CERs), were identified as key players in lipid-induced insulin resistance, underlying type 2 diabetes (T2D). Furthermore, recent results suggest that the subcellular distribution of such lipids strongly affects their diabetogenic properties. We developed a novel LC-MS/MS method for the rapid and simultaneous quantification of DAG and CER species in subcellular fractions of skeletal muscle samples. In total, 12 DAG and 7 CER species can be analyzed simultaneously by multiple reaction monitoring (MRM) within 15 min after standard lipid extraction and solid phase extraction. Quantification is achieved by addition of isotope labeled standards prior to lipid extraction. The assay is characterized by excellent recovery of analytes (DAGs > 81,3%; CERs > 83%), limit of quantification (LOQ; DAGs 3,9 – 4,9 fmol; CERs 1,9 – 2,5 fmol), accuracy (DAGs 104,5 ± 7,7%; CERs 108,2 ± 13,2%) and precision (coefficient of variation, CV; DAGs < 11%; CERs < 9%). We established a fractionation protocol that allows the separation of subcellular fractions (membranes, cytosol and lipid droplets) in tissue samples by differential centrifugation. As an example, the concentration of DAGs and CERs was quantified in subcellular fractions of human muscle samples (CV; DAGs < 18,5% and CERs < 10,5%). In conclusion, this method presents a valuable tool for future human studies to analyze the subcellular dynamics of lipotoxins underlying the pathogenesis of insulin resistance and T2D.